Our HIV vaccine approach is based on initial immunization with a replicating adenovirus (Ad) vector carrying an HIV gene(s) followed by a booster immunization with an HIV envelope protein. The Ad-HIV vaccine replicates in epithelial cells that line mucosal inductive sites, thus eliciting strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We have shown that initial immunizations with an Ad-HIV vaccine also stimulates production of anti-HIV antibodies. Together, the regimen induces strong and durable protective responses. We have demonstrated that administration of Ad-HIV vaccine to the upper respiratory tract as well as to the gut (by oral immunization) elicits memory T cells that possess "homing receptors" leading them to traffic to the intestine, a prime site of HIV infection. Thus the vaccine approach elicits cellular immunity at a location critical for preventing or controlling HIV infection. We are exploring the detection of homing receptors following vaccination as surrogate markers of cellular mucosal immunity. Such surrogates will be invaluable when large scale clinical vaccine trials are carried out. It will not be possible to obtain tissue biopsies from such large numbers of volunteers, and surrogate markers that can be measured on blood samples will be critical in evaluating the development of mucosal immune responses. Currently, we are investigating several alternative immunization routes in a rhesus macaque model including intrarectal, intravaginal, and sublingual. These studies will determine which one elicits optimal immune responses in blood as well as at gastrointestinal and genital/rectal sites, and thus should be translated to future clinical trials. The study of NK cells, key effector cells of innate immunity, is also on-going in order to elucidate their response to Ad-HIV vaccine immunization and their cooperation with vaccine-elicited antibodies in mediating cell killing functions such as antibody-dependent cellular cytotoxicity and antibody-dependent cell mediated viral inhibition. Studies have shown that these activities that span innate and adaptive immunity are correlated with reduced viremia following viral challenge of immunized rhesus macaques. Expanded analyses of polyfunctional memory T cells by intracellular cytokine staining and multi-color flow cytometry analysis are also aimed at improving vaccine design. Finally, we have established methodology to evaluate secretion of a large panel of cytokines and chemokines in response to the Ad-recombinant prime/envelope protein boost regimen. This analysis will further elucidate the host response to the vaccine regimen, providing basic information essential for continued optimization of the vaccine strategy.